The present invention relates to spiro[cyclopent[b]indole-piperidines]. More particularly, the present invention relates to spiro[cyclopent[b]indole-piperidines] of formula 1 
wherein X is hydrogen, halogen, loweralkoxy, loweralkyl, hydroxy, trifluoromethyl and m is 1 or 2 or a group of the formula 
wherein R is loweralkyl and R3 is hydrogen or loweralkyl; R1 is hydrogen or loweralkyl; R2 is hydrogen, a group of the fornula 
wherein n is 1 or 2 and X and m are as above, a group of the formula 
wherein X and m are as above, or a group of the formula 
wherein X and m are as above, Y is hydrogen, or a group of the formula 
wherein R4 is hydrogen or loweralkyl and p is 2 or 3; the optical isomers thereof; or the pharmaceutically acceptable acid addition salts thereof useful in relieving memory dysfunction and thus indicated in the treatment of Alzheimer""s disease, as well as useful in the treatment of depression.
Subgeneric to the compounds of formula 1 are those wherein R2 is hydrogen or a group of the formula 
The present invention relates to (Nxe2x80x2-phenyl)hydrazones of formula 2 
wherein R2 is hydrogen or loweralkyl; R5 is hydrogen or loweralkyl; and X is hydrogen, halogen, loweralkoxy, loweralkyl, hydroxy or trifluoromethyl and m is 1 or 2; the optical isomers thereof; or the pharmaceutically acceptable salts thereof, useful as intermediates for the preparation of the spiro[cyclopent[b]indole-piperidines] of formula 1 and also for the treatment of depression, and 4-yanopiperidines of formula 3 
wherein R6 is loweralkyl; and R7 is halogen or cyano, useful as intermediates for the preparation of the spiro[cyclopent[b]indole-piperidines] of formula 1.
As used throughout the specification and appended claims, the term xe2x80x9calkylxe2x80x9d refers to a straight or branched chain hydrocarbon radical containing no saturation and having 1 to 8 carbon atoms. Examples of alkyl groups are methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 1-hexyl, 3-hexyl, 4-heptyl, 2-octyl and the like. The term xe2x80x9calkoxyxe2x80x9d refers to a monovalent substituent which consists of an alkyl group linked through an ether oxygen having its free valence bond from the ether oxygen. Examples of alkoxy groups are methoxy, ethoxy, propoxy, 1-butoxy, 1-pentoxy, 3-hexoxy, 4-heptoxy, 2-octoxy and the like. The term xe2x80x9calkanolxe2x80x9d refers to a compound formed by a combination of an alkyl group and hydroxy radical. Examples of alkanols are methanol, ethanol, 1- and 2-propanol, 2,2-dimethylethanol, hexanol, octanol and the like. The term xe2x80x9chalogenxe2x80x9d refers to a member of the family fluorine, chlorine, bromine, or iodine. The term xe2x80x9clowerxe2x80x9d as applied to any of the aforementioned groups refers to a group having a carbon skeleton containing up to and including 6 carbon atoms.
The compounds of the present invention which lack an element of symmetry exist as optical antipodes and as the racemic forms thereof. The optical antipodes may be prepared from the corresponding racemic forms by standard optical resolution techniques, involving, for example, the separation of diastereomeric salts of those instant compounds characterized by the presence of a basic amino group and an optically active acid, or by synthesis from optically active precursors.
The present invention comprehends all optical isomers and racemic forms thereof of the compounds disclosed and claimed herein and the formulae of the compounds shown herein are intended to encompass all possible optical isomers of the compounds so depicted.
The novel spiro[cyclopent[b]indole-piperidines] of the present invention are prepared by the processes delineated in Reaction Scheme A.
To prepare a spiro[cyclopent[b]indole-piperidine] of formula 1 an 8-azaspiro[4.5]decane-1-one 4 is condensed with a phenylhydrazine of formula 5 
wherein R1 is hydrogen or loweralkyl and X and m are as above to provide a phenylhydrazone of formula 2 wherein R1, X and m are as above, which is cyclized to a spiro[cyclopent[b]indole-piperidine] 1 wherein R1, X and m are as above and R2 is hydrogen. The condensation is carried out by treating the cyclopentanone 4 with a phenylhydrazine 5 in the presence of an organic carboxylic acid such as acetic acid or a mineral acid such as hydrochloric acid in an alkanol such as ethanol at an elevated temperature such as that within the steam bath range to provide 2.
The cyclization is accomplished by treating a phenylhydrazone 2 with an aqueous alkanolic mineral acid such as ethanolic hydrogen chloride at a temperature within the steam bath range to provide 1.
To fabricate a 1xe2x80x2-benzoylspiro[cyclopent[b]indole-piperidine] 1 wherein R1 is lowerakyl, R2 is 
and X, m and n are as above or a 1xe2x80x2-phenylethylspiro[cyclopent[b]indole-piperidine] 1 wherein R1 is loweralkyl and R2 is 
wherein X, m and n are as above, a spiro[cyclopent[b]indole-piperidine] 1 wherein R1 is loweralkyl, R2 is hydrogen and X and m are as above is treated with a benzoyl halide of formula 6 
wherein X and m are as above and Hal is bromo or chloro or a pnenylalkyl halide of formula 7 
wherein X, m, n and Hal are as above in the presence of a triloweralkylamine such as triethylamine in a halocarbon solvent such as dichioromethane at a temperature within the range from about 0xc2x0 C. to about ambient temperature, or an alkali metal carbonate such as potassium carbonate in an organic solvent such as acetonitrile at a temperature of about the reflux temperature of the reaction medium.
Similarly, a 1xe2x80x2-phenoxyalkylspiro[cyclopent[b]indole-piperidine] 1 wherein R1 is loweralkyl and R2 is a group of the formula 
wherein X and m are as above, Y is hydrogen or a group of the formula 
wherein R4 is hydrogen or loweralkyl and p is 2 or 3 is prepared by treating a spiro[cyclopent[b]indole-piperidine] 1 wherein R1 is hydrogen or loweralkyl, R2 is hydrogen and X and m are as above with a phenoxyalkyihalide of formula 8 
wherein X, Y, m, p and Hal are as above and an inorganic base such as cesium carbonate in an organic solvent such as acetonitrile at a temperature of about 80xc2x0 C.
The preparation of the starting material, 8-azaspiro[4,5]decane-1-one 4, for the elaboration of the spiro [cyclopentd indole-piperidines] of the present invention is outlined in Reaction Scheme B and exemplified in the Examples. In this process, commercially available 4-acetamiddopiperidine 9 is acylated to N-ethoxycarbonyl-4-acetamidopiperidine 10, which in turn is converted to 4-cyano-N-ethoxycarbonylpiperdine 11 and then alkylated to 4-(3-chloropropyl)-4-cyano-N-ethoxycarbonylpiperidine 12. 4-(3-Chloropropyl)4-cyano-N-ethoxycarbonylpiperidine 12 is converted to 4-cyano-4-(3-cyanopropyl)-N-ethoxycarbonyl piperidine 13, which is cyclized to 4-cyano-1-imino-8-azaspiro[4,5]decane 14 and hydrolyzed to 1. While the process for the synthesis of the starting material 4 for the preparation of the ultimate spiro[cyclopent[b]indole-piperidines] 1 is illustrated with N-ethoxycarbonylpiperidines (10 to 14), the scheme is equally applicable for N-loweralkoxycarbonylpiperidines. 
The spiro[cyclopent[b]indole-piperidines] and related compounds of the present invention are useful as agents for the relief of memory dysfunction, particularly dysfunctions associated with decreased cholinergic activity such as those found in Alzheimer""s disease. Relief of memory dysfunction activity is demonstrated in the in vitro inhibition of acetylcholinesterase assay, an assay for the determination of the ability of a drug to inhibit the inactivation of acetylcholine, a neurotransmitter implicated in the etiology of memory dysfunction and Alzheimer""s dementia. In this assay, a modification of a test described by G. L. Ellman, et al., Biochemical Pharmacology, 7, 88 (1961), the following reagents are prepared and employed:
1. 0.005M Phosphate Buffer (pH 7.2)
A solution of monobasic sodium phosphate monohydrate (6.85 g) in distilled water (100 ml) is added to a solution of dibasic sodium phosphate heptahydrate (13.4 g) and distilled water (100 ml) until a pH of 7.2 was attained. The solution was diluted 1 to 10 with distilled water.
2. Substrate in Buffer
The 0.05M Phosphate Buffer (pH 7.2) was added to acetylthiocholine (198 mg) to a total volume of 100 ml, i.e., a quantity sufficient (gs) to 100 ml.
3. 5,5-Dithiobisnitrobenzoic Acid in Buffer
The 0.05M Phosphate Buffer (pH 7.2) was added to 5.5-dithiobisnitrobenzoic acid to a total volume of 100 ml, i.e., a quantity sufficient (gs) to 100 ml.
4. Stock Solution of Drug
A 2 millimolar stock solution of the test drug is prepared in a quantity sufficient of either acetic acid or dimethyl sulfoxide to volume with 5,5-dithiobisnitrobenzoic acid in Buffer. Stock Solution of Drug is serially diluted (1:10) so that the final cuvette concentration is 10xe2x88x924 molar.
Male Wistar rats are decapitated, brains rapidly removed, corpora striata dissected free, weighted and homogenized in 19 volumes (approximately 7 mg protein/ml) of 0.05M Phosphate Buffer (pH 7.2) using a Potter-Elvejhem homogenizer. A 25 xcexcl aliquot of this suspension is added to 1 ml of the vehicle or various concentrations of the test drug and preincubated for 10 minutes at 37xc2x0 C. Enzyme activity is measured with a Beckman DU-50 spectrophotometer with the following software and instrument settings:
1. Kinetics Soft-Pac(trademark) Module #598273;
2. Program #6 Kindata;
3. Sourcexe2x80x94Vis;
4. Wavelengthxe2x80x94412 nm;
5. Sipperxe2x80x94none;
6. Cuvettesxe2x80x942 ml cuvettes using auto 6-sampler;
7. Blankxe2x80x941 for each substrate concentration;
8. Interval timexe2x80x9415 seconds (15 or 30 seconds for kinetics);
9. Total timexe2x80x945 minutes (5 to 10 minutes for kinetics);
10. Plotxe2x80x94yes;
11. Spanxe2x80x94autoscale;
12. Slopexe2x80x94increasing;
13. Resultsxe2x80x94yes (gives slope); and
14. Factorxe2x80x941.
Reagents are added to the blank and sample cuvettes as follows:
1. Blank:
0.8 ml 5xe2x80x945-Dithiobisnitrobenzoic Acid
0.8 ml Substrate in Buffer
2. Control: 0.8 ml 5.5-Dithiobisnitrobenzoic Acid/Enzyme
0.8 ml Substrate in Buffer
3. Drug: 0.8 ml 5.5-Dithiobisnitrobenzoic Acid/Drug/Enzyme
0.8 ml Substrate in Buffer
Blank values are determined for each run to control for non-enzymatic hydrolysis of substrate and these values are automatically subtracted by the Kindata program available on kinetics soft-pac module. This program also calculates the rate of absorbance change for each cuvette.
Substrate concentration is 10 millimolar diluted 1:2 in assay yielding final concentration of 5 millimolar. 5,5-dithiobisnitrobenzoic acid concentration is 0.5 millimolar yielding 0.25 millimolar final concentration.       %    ⁢          xe2x80x83        ⁢    Inhibition    =                              Slope          ⁢                      xe2x80x83                    ⁢          Control                -                  Slope          ⁢                      xe2x80x83                    ⁢          drug                            Slope        ⁢                  xe2x80x83                ⁢        Control              xc3x97    100  
IC50 values are calculated from log-probit analysis
Relief of memory dysfunction is achieved when the present spiro[cyclopent[b]indole-piperidines] and related compounds are administered to a subject requiring such treatment as an effective oral, parenteral or intravenous dose of from 0.10 to 50 mg/kg of body weight per day. A particularly effective amount is about 10 mg/kg of body weight per day. It is to be understood, however, that for any particular subject, specific dosage regimens should be adjusted according to the individual need and the professional judgment of the person administering or supervising the administration of the aforesaid compound. It is to be further understood that the dosages set forth herein are exemplary only and that they do not, to any extent, limit the scope or practice of the invention.
The spiro[cyclopent[b]indole-piperidines] of the present invention are also useful as agents for treating depression. Depression treatment is demonstrated in the in vitro inhibition of monoamine oxidase assay, an assay for the determination of the ability of a drug to inhibit the enzyme monoamine oxidase. In this assay, a modification of an assay described by M. V. Kindt, et al., Europ. J. Pharmnacol. 146: 313-318 1988.
The following reagents are prepared:
1. Phosphate buffer (0.5 M), pH 7.4; 134.4 g dibasic sodium phosphate heptahydrate q.s. to 1 liter in distilled water (A) 17.3 g monobasic sodium phosphate q.s. to 250 ml in distilled water (B) Adjust pH of A to 7.4 by slowly adding B (volumes as needed) Dilute 1:10 in distilled water (0.05 M phosphate buffer, pH 7.4)
2. 0.25 M Sucrose (phosphate buffered): 21.4 g sucrose, q.s. to 250 nil with 0.05 M phosphate buffer
3. Substrate for monoamine oxidase-A:
a. serotonin creatine sulphate (5-hydroxytryptamine) is obtained from Sigma Chemical Company. A 5 mM stock solution ig made up in 0.01 N-hydrochloric acid. The solution is used to dilute the specific activity of the [3H]-hydroxytryptamine.
b. [3H]-5-hydroxytryptamine binoxalate (20-30 Ci/mmol) is obtained from New England Nuclear.
c. Add 12 xcexcl of [3H]-5-hydroxytryptamine to 2 ml of the 5 mM 5-hydroxytryptamine solution. (Final amine concentration in the assay is 200 xcexcM: see below.)
4. Substrate for monoamine oxidase-B
a. xcex2-phenethylamine is obtained from Sigma Chemical Company. A 5 mM stock solution is made up in 0.01 N-hydrochloric acid. The solution is used to dilute the specific activity of the [14C]-xcex2-phenethylamine.
b. xcex2-[ethyl-1-14C]-phenethylamine hydrochloride (40-50 mCi/mmol) is obtained from New England Nuclear.
c. Add 12 xcexcl of [14C]-xcex2-phenothylaamine to 2 ml of the 5 mM xcex2-phenethylamine solution. (Final amine concentration in the assay is 200 xcexcM: see below.)
5. Equal amounts of monoamine oxidase-A (5-hydroxyttyptamine) and monoamine oxidase-B (xcex2-phenethylamine) substrates are combined for simultaneously testing both monoamine oxidase types, i.e., mixed stock solution of 2.5 mM 5-hydroxytryptamine and 2.5 mM xcex2-phenethylamine. 40 xcexcl of this mixed solution gives a 200 xcexcM final concentration of each amine in the assay. When testing only one monoamine oxidase type, the individual 5 mM stock solutions must be diluted 1:1 with distilled water prior to adding 40 xcexcl to the incubation mixture; i.e., same 200 xcexcM final amine concentration.
6. Stock solutions of test drugs are made up in appropriate vehicles and serially diluted to give final concentrations ranging from 10xe2x88x927 to 10xe2x88x923 molar in the assay. Lower concentrations can be made for more potent drugs.
Tissue Preparation
Male Wistar rats weighing 150-250 grams were sacrificed and the brains rapidly removed. Whole brain minus cerebellum was homogenized in 30 volumes of ice-cold, phosphate-buffered 0.25 M sucrose, using a Potter-Elvejhem homogenizer. The homogenate was centrifuged at 1000 g for 10 minutes and the supernatant (S1) decanted and recentrifuged at 18,000 g for 20 minutes. The resulting pellet (P2) was resuspended in fresh 0.25 M sucrose and serves as the tissue source for mitochondrial monoamine oxidase.
C. Assay
10 xcexcl 0.5 M phosphate buffer, pH 7.4
50 xcexcl water or appropriate drug concentration
400 xcexcl tissue suspension
Tubes are preincubated for 15 minutes at 37xc2x0 C. and the assay is started by adding 40 xcexcl of combined substrate ([3H]-5-hydroxytryptamine and [14C]-xcex2-phenethylamin) at 15 second intervals. The tubes are incubated for 30 minutes at 37xc2x0 C. and the reaction stopped by the addition of 0.3 ml 2N-hydrochloric acid. Tissue blank values are determined by adding the acid before the radioactive substrate. The oxidative products of the reaction are extracted with ethylacetate/toluene (1:1). Add 5 mnl of this mixture to the tubes, vortex for 15 seconds to extract the deaminated metabolites into the organic phase and allow to separate from the aqueous phase. Place tubes in acetone/dry ice bath to freeze the aqueous layer. When this layer is frozen, pour off the top organic layer into a scintillation vial. Add 10 ml Liquiscint and count the samples using window settings for 14C in one channel and 3H in the second channel. IC50 values are determined by log-probit analysis.
Results
Depression treatment is achieved when the present spiro[cyclopent[b]indole-piperidines] and related compounds are administered to a subject requiring such treatment as an effective oral, parenteral or intravenous dose of from 0.10 to 50 mg/kg of body weight per day. A particularly effective amount is about 10 mg/kg of body weight per day. It is to be understood, however, that for any particular subject, specific dosage regimens should be adjusted according to the individual need and the professional judgment of the person administering or supervising the administration of the aforesaid compound. It is to be further understood that the dosages set forth herein are exemplary only and that they do not, to any extent, limit the scope or practice of the invention.
Acetylcholinesterase inhibitors and monoamine oxidase inhibitors are known in the art as being useful as relievers of memory dysfunction and as antidepressants, respectively. (See V. Kumar in Alzheimer""s Disease: Therapeutic Strategies, E. Giacobini and R. Becker Eds.; Birkhauser, Boston 1994 for memory dysfunction utility and K F. Tipton in Biochemical and Pharmacological Aspects of Depression, K. F. Tipton and U. B. H. Youdin, Eds., Taylor and Francis, London 1989 for antidepressant utility.
Effective amounts of the compounds of the invention may be administered to a subject by any one of various methods, for example, orally as in capsules or tablets, parenterally in the form of sterile solutions or suspensions, and in some cases intravenously in the form of sterile solutions. The free base final products, while effective themselves, may be formulated and administered in the form of their pharmaceutically acceptable addition salts for purposes of stability, convenience of crystallization, increased solubility and the like.
Preferred pharmaceutically acceptable addition salts include salts of mineral acids, for example, hydrochloric acid, sulfuric acid, nitric acid and the like, salts of monobasic carboxylic acids such as, for example, acetic acid, propionic acid and the like, salts of dibagic carboxylic acids such as, for example, maleic acid, fumaric acid, oxalic acid and the like, and salts of tribasic carboxylic acids such as, for example, carboxysuccinic acid, citric acid and the like.
The active compounds of the present invention may be administered orally, for example, with an inert diluent or with an edible carrier. They may be enclosed in gelatin capsules or compressed into tablet. For the purpose of oral therapeutic adm;nistration, the aforesaid compounds may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. These preparations should contain at least 0.5% of active compounds, but may be varied depending upon the particular form and may conveniently be between 4% to about 75% of the weight of the unit. The amount of present compound in such composition is such that a suitable dosage will be obtained. Preferred compositions and preparations according to the present invention are prepared so that an oral dosage unit form contains between 1.0-300 mgs of active compound.
The tablets, pills, capsules, troches and the like may also contain the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, corn starch and the like; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; and a swcctcning agent such as sucrose or saccharin or a flavoring agent such as peppermint, methyl, salicylate, or orange flavoring may be added. When the dosage unit is a capsule it may contain, in addition to materials of the above type, a liquid carrier such as fatty oil. Other dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings. Thus tablets or pills may be coated with sugar, shellac, or other enteric coating agents. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
For the purposes of parenteral therapeutic administration, the active compounds of the invention may be incorporated into a solution or suspension. These preparations should contain at least 0.1% of the aforesaid compound, but may be varied between 0.5 and about 50% of the weight thereof. The amount of active compound in such compositions is such that a suitable dosage will be obtained. Preferred compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.5 to 100 mgs of the active compound.
The solutions or suspensions may also include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparations can be enclosed in ampules, disposable syringes or multiple vials made of glass or plastic.